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Dominant selectable markers are beneficial for transformation of many fungi, particularly those model species where repeated transformations may be required. A carboxin resistance allele of the Coprinopsis cinerea sdi1 gene, encoding the iron-sulphur protein subunit of succinate dehydrogenase, was developed by introducing a suitable point mutation in the histidine block responsible for binding of the associated iron ion. This modified gene was used successfully to confer carboxin resistance upon transformation of C. cinerea protoplasts. Plasmids previously used to establish hygromycin transformation systems of several basidiomycete species, such as pAN7-1 and phph004, failed to give rise to hygromycin-resistant transformants of C. cinerea, whilst pPHT1 was successful . Sequencing of these constructs showed that the hygromycin resistance gene in pAN7-1 and phph004 had been modified removing the codons encoding two lysine residues following the N-terminal methionine. Replacement of the deleted 6 bp (AAA AAG) in the truncated hph gene led to generation of hygromycin-resistant transformants indicating the importance of these two codons for expression in C. cinerea. Phleomycin-resistant ( ble) transformants were also obtained, but only with the intron-containing construct pblei004, showing that an intron is necessary to obtain phleomycin-resistant C. cinerea. This contrasts with hygromycin-resistance , where introns are not required for expression, emphasising the variability in importance of these elements. [ABSTRACT FROM AUTHOR]

To understand the molecular mechanisms of fruiting body formation of basidiomycetous mushrooms, we have isolated over a 100 of developmentally regulated genes that were specifically transcribed during fruiting body development in Lentinula edodes ( Shiitake-mushroom) by a subtractive hybridization, cDNA-RDA (cDNA representational difference analysis). One of these genes, named Le.flp1, was isolated from the primordial cDNA library of L. edodes, and the expression product of Le.flp1 and putative fungal homologues contained a characteristic region, homologous to the Fas domain of fasciclin family proteins, which are capable of promoting cell adhesion through Fas domain-mediated homophilic interactions in various organisms. RT-PCR analyses suggested that Le.flp1 was specifically expressed in primordia and mature fruiting bodies. In situ hybridization indicated that Le.flp1 transcripts were distributed distinctly in the following tissues: the inside of gills of fruiting bodies, especially at the boundary between the subhymenium and trama, where there is active proliferation of basidium cells for producing basidiospores; peripheral regions of the primordium, pileus and stipe; and both inner tissue and outer regions of the stipe. Our results suggest the hypothesis that Le.flp1 plays a role in cellular differentiation and development in ubiquitous tissues during fruiting body formation in L. edodes, possibly through cell adhesion. [ABSTRACT FROM AUTHOR]

We have isolated and characterised the first set of chitin synthase genes from a rust fungus, a large group of economically highly important, obligately biotrophic plant pathogens. Puccinia graminis was used as a model organism for the rust fungi which are not well investigated on the molecular level today. One of the major structural components of most fungal cell walls is the chitin polymer which is synthesised by a family of enzymes called chitin synthases. In P. graminis, we have isolated five new chitin synthase genes from four different classes, chsII, chsIIIa, chsIIIb, chsIV, and chsV. The genes contain a high number of introns, unusual for other known fungal chitin synthases. The dinucleic stage of the fungus seems to contain two slightly different genes or alleles for four isoforms. One isoform, chsIIIa, seems to be expressed only in the youngest stages of fungal growth. Analysis of the derived proteins shows that together with other basidiomycete CHS, the pgtCHS form separate subgroups in the phylogenetic tree. This set of five rust chitin synthase genes, with some unusual features compared to known fungal chitin synthases, allows new insights into chitin synthase classification, and may help in the development of novel functional fungicides. [ABSTRACT FROM AUTHOR]

The apical extension of hyphae is of central importance for extensive spread of fungal mycelium in forest soils and for effective ectomycorrhiza development. Since the tubulin cytoskeleton is known to be important for fungal tip growth, we have investigated the expression of an α-tubulin gene from the ectomycorrhizal basidiomycete Amanita muscaria ( AmTuba1). The phylogenetic analysis of protein sequences revealed the existence of two subgroups of α-tubulins in homobasidiomycetes, clearly distinguishable by defined amino acids. AmTuba1 belongs to subgroup1. The AmTuba1 transcript level is related to mycelial growth rate. Growth induction of carbohydrate starved (non-growing) hyphae resulted in an enhanced AmTuba1 expression as soon as hyphal growth started, reaching a maximum at highest mycelial growth rate. Bacterium-induced hyphal elongation also leads to increased AmTuba1 transcript levels. In mature A. muscaria/ P. abies ectomycorrhizas, where fungal hyphae are highly branched, and slowly growing, AmTuba1 expression were even lower than in carbohydrate-starved mycelium, indicating a further down-regulation of gene expression in symbiosis. In conclusion, our analyses show that the AmTuba1 gene can be used as a marker for active apical extension in fly agaric, and that α-tubulin proteins are promising tools for the classification of fungi. [ABSTRACT FROM AUTHOR]

In the current studies, we sequenced and characterized the gene for the homeodomain protein ( hox1) in a bipolar mushroom, Pholiota nameko, which is a putative homologue of A mating type genes in the tetrapolar basidiomycete, Coprinopsis cinerea. We also sequenced and characterized the gene for the pheromone receptor ( rcb1) in P. nameko, which is a putative homologue of the B mating type genes in C. cinerea. Restriction fragment length polymorphism (RFLP) and linkage analyses indicated that the both genes are present as a single locus on the different chromosome. Moreover, in P. nameko, the hox1 gene was mapped to the A mating type locus in linkage group I. However, rcb1 was not linked to the A mating type locus and was mapped to the other linkage group. These results strongly suggest that hox1 regulates with incompatibility in the bipolar mushroom, and that rcb1 may not affect the mating function in P. nameko. This is the first report regarding the structure of the mating type genes in bipolar mushrooms. [ABSTRACT FROM AUTHOR]